Appropriate regulation of neuronal function, such as synaptic plasticity, depends in part on neurotransmitter receptors and ion channels, especially those localized in synapses. Phosphorylation/ dephosphorylation of serines and/or threonines in the receptor/ion channel subunits is a major mechanism for their regulation; precise modulation of protein kinase and protein phosphatase activities is therefore essential for learning and memory. For example, activation of type 1 protein phosphatase catalytic subunit (PP1C) isoforms localized in dendritic spine sis likely important in the induction of long-term depression. We characterized four rat brain cytoskeletal, isoform- selective, PP1C-binding proteins (PP1bps) and have purified an actin- associated PP1 holoenzyme (PP1A) containing PP1bp134, PP1bp175 and PP1C. We hypothesize that association of PP1C with these PP1bps specifically targets and regulates neuronal PP1 activity. Three complementary Specific Aims are proposed, which will identify PP1bp134 and PP1bp175 and begin to characterize their physiological role: 1. Characterization and cloning of PP1bps. The cDNAs encoding PP1bps present in PP1A (PP1bp134, PP1bp175) will be isolated. The authenticity of cDNAs will be verified by expression of PP1bp activity in heterologous systems, and by immunological identification of the cloned proteins in PP1A. Expression patterns of PP1C isoform and novel PP1bp mRNAs in tissues, brain regions and during development will be compared. 2. Characterization of the interaction of PP1C, with PP1bps. A. Domain interactions in vitro: Deletion, truncation, and site-directed PP1bp mutants (expressed in bacteria) will be used to identify the minimal isoform selective PP1C binding domain using multiple binding assays. B. Interactions in vivo: Interaction of PP1bps with PP1C isoforms in brain region extracts, brain slices and cultured neurons will be elucidated by co-immunoprecipitation, immunoblotting subcellular fractions, and immunofluorescent confocal microscopy. Effects of wild-type and mutated PP1bps on PP1C localization in undifferentiated heterologous cells (e.g., HEK293, NG108, PC12) and during neuronal differentiation, and on neuronal differentiation itself, will be investigated. 3. Regulation of PP1A. The regulation of PP1bp binding to PP1C and actin by Ca2+/calmodulin-dependent protein kinase II will be investigated and specific phosphorylation sites will be identified. Phosphorylation of PP1bps will be examine din intact cells/neurons and the effects on PP1C/PP1bp localization will be elucidated.
These Aims provide novel insights into the physiological regulation of PP1, a key modulator of synaptic function.
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